Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing

Abstract

Rationale: Individuals with naturally occurring loss-of-function proprotein convertase subtilisin/kexin type 9 (PCSK9) mutations experience reduced low-density lipoprotein cholesterol levels and protection against cardiovascular disease.

Objective: The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system can efficiently introduce loss-of-function mutations into the endogenous PCSK9 gene in vivo.

Methods and results: We used adenovirus to express CRISPR-associated 9 and a CRISPR guide RNA targeting Pcsk9 in mouse liver, where the gene is specifically expressed. We found that <3 to 4 days of administration of the virus, the mutagenesis rate of Pcsk9 in the liver was as high as >50%. This resulted in decreased plasma PCSK9 levels, increased hepatic low-density lipoprotein receptor levels, and decreased plasma cholesterol levels (by 35-40%). No off-target mutagenesis was detected in 10 selected sites.

Conclusions: Genome editing with the CRISPR-CRISPR-associated 9 system disrupts the Pcsk9 gene in vivo with high efficiency and reduces blood cholesterol levels in mice. This approach may have therapeutic potential for the prevention of cardiovascular disease in humans.

Keywords: coronary disease; genetic therapy; lipoproteins; molecular biology; prevention and control.

Figure 1. On-target and off-target effects in mouse cells and livers receiving CRISPR-Cas9

A shows Surveyor assays performed with genomic DNA from 3T3-L1 cells transfected with Cas9 and a guide RNA targeting Pcsk9 exon 1 (gRNA-1) or a guide RNA targeting Pcsk9 exon 2 (gRNA-2). B shows Surveyor assays performed with genomic DNA from liver samples taken from mice three days after receiving a control adenovirus expressing GFP (A, B) or an adenovirus expressing Cas9 and gRNA-1 (CRISPR-Pcsk9) (C, D). C shows Surveyor assays performed with genomic DNA from liver samples taken from mice four days after receiving no virus (1–5), the GFP virus (6–10), or the CRISPR-Pcsk9 virus (11–15). D shows Surveyor assays performed with liver genomic DNA from the “A” and “C” mice. The Pcsk9 exon 1 on-target site and ten genomic sites deemed to be the most likely off-target sites for CRISPR-Cas9 activity (OT1–OT10; see Online Supplement for site sequences) were assessed. Arrows show the cleavage products resulting from the Surveyor assays; the intensity of the cleavage product bands relative to the uncleaved product band corresponds to the mutagenesis rate.

Figure 2. Effects of CRISPR-Cas9 genome editing on mice

A shows the results of ELISAs for PCSK9 protein, measurements of triglyceride levels, and measurements of total cholesterol levels in plasma samples from mice four days after receiving no adenovirus, GFP adenovirus, or CRISPR-Pcsk9 adenovirus (N = 5 mice for each group). B shows the full plasma lipoprotein cholesterol profiles of pooled plasma samples from each group of mice. C shows the plasma ALT levels in mice four days after receiving virus (N = 5 mice for each group) and hematoxylin/eosin stains of liver sections from representative mice. D shows the results of Western blot analysis of liver samples taken from mice four days after receiving virus. For A and C, P values were determined by the Kruskal-Wallis test among all three groups (in red); if statistically significant, the Mann-Whitney U test between each pair of groups was performed (P values in black). Error bars show s.e.m.